Research was conducted to investigate the biosynthesis of pertussis toxin (PT) as well as the structure/function relationships of the toxin. While the current dogma in the field is that only B. pertussis produces PT, recent clinical trials have provided provocative results which may suggest otherwise. A number of clinical investigators have reported that sera from children infected with either B. parapertussis or B. bronchiseptica can show antibody rises to PT. It has been known for some time that both B. parapertussis and B. bronchiseptica contain ptx genes but the genes were thought to be transcriptionally silent. We asked the question of whether the ptx genes of B. parapertussis and B. bronchiseptica could produce functionally active PT, if the genes were expressed. We constructed strains of both B. parapertussis and B. bronchiseptica which had the B. pertussis ptx promoter inserted immediately in front of the ptx- ptl regions of those strains. We found that both species produced active PT as well as at least certain of the Ptl proteins. The S2 subunit was confirmed to be prematurely terminated in B. parapertussis. Nonetheless, an active toxin was produced, although this toxin appeared to be more labile that PT. These results suggest that, if the ptx-ptl genes of B. parapertussis and B. bronchiseptica were expressed during the disease process, they would be functionally active and might contribute to pathogenesis. Moreover, these data illustrate that a significant number of amino acid changes can occur in PT without destroying biological activity. These results have implications for the future of acellular pertussis vaccines and suggest that we must remain vigilant to ensure that clinical isolates of B. pertussis do not exhibit alteration in PT sequence that might affect the efficacy of acellular pertussis vaccines that contain an inactivated form of this protein. We are currently using genetic and molecular biological techniques to determine whether the ptx genes of B. parapertussis and B. bronchiseptica are ever expressed in vitro or during the course of infection in vivo.